The specific aims of this project are to determine the cellular and molecular mechanisms involved in the regulation of Na-K-C1 cotransport in mammalian (including human) airway epithelial cells, and whether the regulation of this cotransporter is intact in airway cells from patients with cystic fibrosis (CF). Na-K-C1 cotransport is the major pathway for basolateral NaC1 entry into both human and canine airway cells, and the activity of this pathway must be increased simultaneously with apical C1 channel activity during stimulation of net salt and fluid secretion by beta-adrenergic hormones and other secretagogues. Cyclic AMP (cAMP)- mediated stimulation of apical C1 channels does not occur in CF airways, and one potential therapeutic approach to the airway disease in CF is to develop alternative means to activate C1 channels and net salt secretion in these airways. As such, it is important to know if these means are capable of activating basolateral C1 entry via Na-K-C1 cotransport as well as apical C1 channels; it may also be possible to secondarily activate apical C1 channels and net secretion by first stimulating the cotransporter. Cultured canine and human (normal and CF) airway epithelial cells will be employed in this project. [3H]bumetanide binding and tracer ion flux assays will be used to determine Na-K-C1 cotransport and transepithelial ion transport in these cultures, and their responses to potential stimuli including beta- and alpha-adrenergic agonists, changes in cell volume, bradykinin, histamine, ATP, UTP, and A23187 + Ca. It will be determined if stimulation of basolateral cotransport by these treatments represents a primary effect on the cotransporter, or if such stimulation is secondary to activation of apical C1 channels and possibly a resultant decrease in cell volume. Protein kinase and phosphatase inhibitors will be employed to determine the role of phosphorylation reactions in regulation of Na-K-C1 cotransport by both cAMP-dependent and cAMP-independent mechanisms, and whether different kinases and phosphatases are involved in regulation of apical C1 channels and basolateral cotransporters. Using a photoaffinity label for the Na-K-C1 cotransporter and specific antibodies directed against this transporter, it will be determined if the cotransporter itself is phosphorylated in response to stimuli of net C1 secretion in airway epithelia, and if changes in phosphorylation are correlated with changes in ion transport and [3H]bumetanide binding.